(A) Schematic of T. brucei BES-specific TAR cloning vector pEB4 constructed from the general TAR vector pEB2 (M. Becker and E.J. Louis, unpubl.). The 560-bp T. brucei TAR target including the BES promoter is indicated as the black box labeled TAR. S. cerevisiae telomere repeats are indicated as black arrows. Further indicated are the yeast centromere and the origin of replication (striped box labeled CEN, ARS), the positive selectable marker gene URA3 (grey box labeled URA3) and the negative selectable marker CYH2 (white box labeled CYH2). Restriction enzyme sites ClaI, used for linearization of the vector prior to transformation; ScaI, used for proper orientation of the TAR fragment; and XhoI/SalI, used for insertion of the TAR fragment, are indicated. The latter were destroyed upon insertion. (B) Schematic for selective isolation of telomeric T. brucei bloodstream form VSG expression sites (BES) via TAR cloning in yeast. At the top of the figure is a schematic of a telomeric BES with duplicated promoters indicated with white flags, various expression site-associated genes (ESAGs) indicated with numbered grey boxes, and the VSG with a black box. Characteristic 50-bp and 70-bp repeat arrays are indicated with hatched boxes, and the T. brucei telomere repeats are indicated with white triangles. The linear S. cerevisiae TAR cloning vector pEB4 is indicated below, with S. cerevisiae telomere repeats indicated with black triangles. The 560-bp T. brucei TAR target fragment, including the BES promoter, is indicated with a white box marked TAR. The TAR vector further includes yeast centromere and ARS sequences (black boxes marked CEN/ARS), a positive marker gene URA3 (grey box labeled M), and a negative marker gene CYH2 (grey box labeled CYH). (a) Product of a recombination between the T. brucei target and an upstream BES promoter, producing a linear telomere clone as half YAC. (b) Product of a recombination between the T. brucei target and a downstream BES promoter, producing an ∼13-kb-shorter linear telomere clone.
